Rapid degradation of halogenated hydrocarbons by soluble methane monooxygenase

ABSTRACT

A method is disclosed for degradation of a halogenated hydrocarbon compound such as trichloroethylene (TCE) which utilizes a soluble methane monooxygenase or a bacterium comprising the monooxygenase. Methylosinus trichosporium OB3b is a soluble methane monooxygenase-producing bacterium which when cultivated by continuous culturing comprising exposing the bacterium to a continuous-flow gas mixture of air and methane in a ratio of about 25:1-1:20, respectively. Methylosinus trichosporium OB3b is capable of degrading TCE at rates from about 500-10,000 micromoles per hour per gram cells. The present method is useful to degrade halogenated hydrocarbon compounds such as TCE at initial concentrations up to 10,000 micromoles/l.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The invention described herein was made with the assistance of theNational Institute of General Medical Sciences, National Institutes ofHealth Grant Nos. GM40466 and GM24689, and by a grant from the Centerfor Biological Process Technology of the University of Minnesotasponsored by the National Science Foundation. The Government has certainrights in the invention.

This is a Continuation of Ser. No. 07/886,608 filed May 20, 1992, nowabandoned which is a divisional application of Ser. No. 07/814,202,filed Dec. 20, 1991, now U.S. Pat. No. 5,196,339, which is aContinuation of Ser. No. 07/384,859, filed Jul. 21, 1989, now abandoned,which in turn is a Continuation of Ser. No. 07/272,538, filed Nov. 17,1988, now abandoned.

FIELD OF THE INVENTION

This invention relates to methods of biologically degrading halogenatedhydrocarbon compounds including trichloroethylene (TCE), wherein saidmethods utilize a soluble methane monooxygenase or the methane-oxidizingbacterium which comprises the soluble methane monooxygenase.

BACKGROUND OF THE INVENTION

Halogenated hydrocarbon compounds are high-volume products of thechemical process industry; for example, more than 6 million metric tonsof trichloroethylene (TCE), tetrachloroethylene (PCE), trichloroethane,carbon tetrachloride (CT), and chloroform (CF) are produced (in theUnited States) each year. Those halogenated hydrocarbon compounds mostfrequently found in groundwater are low molecular weight aliphatichalogenated hydrocarbons: TCE, dichloroethane (DCA), trichloroethane,and PCE. Many of these aliphatic halogenated hydrocarbon compounds,including TCE, have been listed as priority pollutants by the U.S.Environmental Protection Agency, and are known or suspected carcinogensand mutagens. Haloforms (halogenated derivatives of methane) are alsofrequently detected in groundwaters and drinking waters. Some haloformsare produced during chlorination of water supplies, but inadequatedisposal techniques or accidental spillage may also be responsible forthe presence of these haloforms.

Several of the halogenated hydrocarbon compounds mentioned above areresistant to biodegradation in aerobic subsurface environments, or theirbiological transformations are incomplete under anaerobic conditions.For example, under anaerobic conditions, TCE and PCE are known toundergo partial bioconversion to vinyl chloride, a compound which is asmuch or more of a problem as the original contaminants. Wilson andWilson, Appl. Env. Microbiol., 49:242-243 (1985).

Current technology for reclaiming groundwater polluted with thesehalogenated hydrocarbon compounds involves pumping water to the surfaceand stripping out the contaminants in aeration towers, or removing thepollutants on a sorbent. The former process is not permitted in somestates, and the latter is expensive and involves the production ofconcentrated toxic materials that may present future problems.

In an alternative reclamation method, acetate-degrading methanogenicbacteria have been reported to degrade halogenated hydrocarboncompounds. Chloroform (CF), bromodichloromethane (BDCM),dibromochloromethane (BDCM), bromoform (BF), carbon tetrachloride (CT),1,1,1-trichloroethane (1,1,1-TCA), 1,1,2,2-tetrachloroethane(1,1,2,2-TECE), and PCE have all been substantially degraded undermethanogenic conditions utilizing an anaerobic column with acetateemployed as the primary substrate in a medium seeded with a methanogenicmixed bacterial culture. A continuous-flow, fixed-film laboratory scalecolumn operated under these conditions with a 2-day retention timesubstantially removed these compounds present at column influentconcentrations ranging from about 15-40 μg/l. The acclimation periodrequired for significant removal of CF, 1,1,1-TCA, and 1,1,2,2-TECE wasabout 10 weeks. Bouwer and McCarty, Appl. Env. Microbiol., 45:1286-1294(1983).

Other anaerobic bacteria are also known to degrade halogenatedhydrocarbon-containing compounds. For example, the anaerobic bacteriaMethanobacterium thermoautotrophicum and D. autotrophicum have beenshown to convert carbon tetrachloride to di- and trichloromethane, andto partially dehalogenate other chlorinated aliphatic compounds. Egli etal., FEMS Microbiol. Letter, 43:257-261 (1987). The above resultsindicate that the use of methanogenic or other anaerobic bacteria tocompletely degrade all halogenated hydrocarbons is not commerciallyviable. These organisms exhibit slow rates of halogenated hydrocarbondestruction, even at low initial concentrations of the hydrocarbons, andare difficult to work with given that anaerobic conditions are required.

Additionally, chloroform is oxidized at rates of 35 nano-moles per gramof cells per minute by the aerobe Methylococcus capsulatus Bath. Higginset al., Nature, 286:561-564 (1980); Haber et al., Science, 221:1147-1153(1983). Similar rates of degradation were observed for other haloformsexcept for carbon tetrachloride, which was not oxidized by Methylococcuscapsulatus Bath. Higgins et al., supra; Haber et al., supra.

Certain methane-oxidizing bacteria are known to degrade chlorinatedhaloforms and halogenated hydrocarbon compounds. For example, soilcolumns exposed to a surface mixture of 0.6% natural gas (primarilymethane) in air for 3 weeks, and having water containing TCE at anaverage concentration of 150 μg/l added to the column influent at theend of the 3-week acclimation period, resulted in less than 5% of theapplied TCE passing through the soil. Wilson and Wilson, supra. Amethane-utilizing mixed culture isolated from a marsh has also recentlybeen shown to completely oxidize TCE, vinyl chloride, vinylidenechloride, and dichloroethylene to carbon dioxide. Fogel et al., Appl.Env. Microbiol., 51:720-724 (1986). However, the rate of TCE degradationreported by Fogel et al. was very slow, approximately 2.5 μmoles perhour per gram of cells. Additionally, tetrachloroethylene was notoxidized by the mixed culture.

The above studies indicate that several chlorinated haloforms andhalogenated hydrocarbon compounds are degradable by combinedaerobic/anaerobic incubation under the proper conditions. However, thereal potential of methane-oxidizing bacteria, or methanotrophs, forrapidly biodegrading halogenated hydrocarbon compounds such as TCE hasnot yet been exploited. For example, when TCE was added to the soilcolumn used by Wilson and Wilson, supra, the soil had previously beenacclimated to the natural gas mixture for 3 weeks. Similarly, theacclimation period required for significant removal of 1,1,1-TCA and1,1,2,2 TECE in the Bouwer and McCarty study was about 10 weeks.

It has been known for some time that obligate methanotrophs derive noenergy from metabolism of compounds other than methane. Haber et al.,supra; Higgins et al., supra. However, methanotrophs are able to degradenumerous hydrocarbon compounds. The ability of methanotrophs to oxidizea wide range of compounds has been associated with the lack ofspecificity of methane monooxygenase (MMO), an enzyme produced bymethanotrophs. Haber et al., Supra; Higgins et al., Supra. The MMOsystem of methanotrophic bacteria catalyzes the cleavage of O₂ andincorporation of one oxygen atom into methane to produce methanol.

The MMO system of methanotrophic bacteria can exist in either a solubleor a particulate (i.e., membrane-bound) form, depending on growthconditions. Burrows et al., J. Gen. Microbiol., 130:3327-3333 (1984),reported that copper availability during the growth of themethanotrophic bacterium Methylosinus trichosporium OB3b (Mt OB3b)determined the intracellular location of its MMO (i.e., whether MMOactivity was located in the particulate or the soluble fraction of thebacterium). However, the tendency of methanotrophic bacteria cells toelaborate only the membrane-bound (particulate) form of MMO has been arecurring problem in the purification of soluble MMO in quantity. Foxand Lipscomb, Biochem. and Biophys. Res. Comm., 154:165-170 (1988).Burrows et al., supra, reported that the particulate form of the MMO ofMt OB3b differed from the soluble form of the enzyme in that theparticulate MMO was unable to oxidize aromatic or alicyclic hydrocarboncompounds. Both the particulate and soluble forms of the MMO of Mt OB3bwere shown to oxidize methane, propene, and various n-alkanes.

To date, however, no one has fully exploited the degradation ability ofmethanotrophic bacteria, nor in particular the degradation ability ofthe soluble form of the MMO produced by these bacteria, in order to bothrapidly and completely degrade halogenated hydrocarbon compounds. Forexample, the rates of TCE degradation by methanotrophic bacteriareported thus far are unsatisfactorily slow and thus impractical forcommercial use. Rates of TCE degradation reported under optimalconditions barely exceed 100 μmoles per hour per gram of cells. Fogel etal., supra; Nelson et al., App. Env. Microbiol., 54:604-606 (1988);Nelson et al., App. Env. Microbiol., 52:383-384 (1986). The time courseof methanotrophic attack upon TCE reported in past studies suggests thatTCE is in some way toxic to the bacteria cells, or to the enzymesfunctional in TCE degradation.

Accordingly, there is a need for a method to rapidly and completelydegrade halogenated hydrocarbon compounds such as TCE by employing thesoluble form of MMO, or by employing a methanotrophic bacterium whichhas been cultured in such a way as to produce the soluble MMO.

SUMMARY OF THE INVENTION

The present invention provides a method of microbial degradation of ahalogenated hydrocarbon compound. The method comprises contacting thehalogenated hydrocarbon compound with an amount of a methane-oxidizingbacterium effective to completely degrade halogenated hydrocarboncompounds such as TCE at a rate from about 500 to about 10,000micromoles per hour per gram of cells. The methane-oxidizing bacteriumis cultured under continuous culture conditions in which the bacteriumis exposed to a continuous-flow gas mixture of air and methane in aratio of about 25:1-1:10, respectively. The continuously culturedbacterium produces a soluble methane monooxygenase (MMO).

Preferably, rates of halogenated hydrocarbon degradation according tothe present invention are from about 1,000 to about 9,000 micromoles perhour per gram of cells. In a preferred embodiment we have achieved ratesof TCE degradation of 2000 to 4000 micromoles per hour per gram dryweight of Methylosinus trichosporium OB3b cells. The present inventionprovides for degradation of halogenated hydrocarbons present in initialconcentrations of up to 10,000 micromoles/l and preferably provides fordegradation of halogenated hydrocarbon compounds such as TCE at initialconcentrations from trace amounts of TCE up to about 1,000 micromoles/l.Moreover, the continuously cultured cells produce soluble MMO at celldensities well below the cell densities required in other studies.

Further, in a preferred embodiment, Methylosinus trichosporium cells areemployed in amounts of from about 0.10g/l to about 20g/l, mostpreferably in amounts of from about 0.2 to 2.0 g/l. The air/methanemixture used for continuous culturing can vary. We have found thatpreferably, degradation of TCE is stimulated when methane is present inamounts from about 1 to about 20% of saturation.

The present method is advantageous in that it both rapidly andcompletely degrades halogenated hydrocarbon compounds such as TCE. Thecontinuous culture conditions employed by the present method to culturethe methane-oxidizing bacterium ensure that TCE will be completelydegraded by the bacterium when the concentration of TCE is significant.Additionally, the utilization of these continuous culture conditionsprovides for the generation of a methane-oxidizing bacterium whichproduces sufficient quantities of the soluble form of MMO. Using thecontinuous culture conditions of the present invention the amount of MMOin the cultured cells is from about 5 to 30% of the weight of dry cells.

The present invention also provides a method of cultivating amethane-oxidizing bacterium capable of completely degrading ahalogenated hydrocarbon compound. The method comprises continuouslyculturing a methane-oxidizing bacterium so that the bacterium producessoluble MMO in an amount effective to completely degrade the halogenatedhydrocarbon compound. Continuous culture conditions include exposure toa gas mixture of air and methane in a ratio from about 25:1 to about1:20 preferably from about 10:1 to about 1:2, and most preferably about2.1:1. Preferably, the amount of MMO in the culture cells is from about5% to about 30% of the weight of dry cells

Further provided by the present invention is a method of degrading ahalogenated hydrocarbon compound using methane monooxygenase, the methodcomprising continuously culturing a methane-oxidizing bacterium;separating a soluble methane monooxygenase therefrom; purifying thesoluble methane monooxygenase to yield the purified componentsreductase, component B, and hydroxylase; adding the purified componentsto an aqueous slurry of the halogenated hydrocarbon compound to form amixture; and reacting the mixture for a period of time sufficient tocompletely degrade the halogenated hydrocarbon compound.

Other features and advantages of the invention will be apparent from thefollowing detailed description and appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphical representation of the time course of TCEdegradation by continuously cultured Mt OB3b, determined at twodifferent values of bacterial cell density.

FIG. 2 is a graphical representation of the time course of TCEdegradation by continuously cultured Mt OB3b in the presence ofdifferent levels of methane.

FIG. 3 shows a chemostat assembly of the type used to grow cultures inaccordance with the present invention.

FIG. 4 shows the head of a chemostat growth flask of the type used togrow cultures in accordance with the present invention.

FIG. 5 shows the body of a chemostat growth flask of the type used togrow cultures in accordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION Halogenated Hydrocarbon-ContainingCompounds

The present invention provides a method of rapidly and completelydegrading a halogenated hydrocarbon compound. Although the presentinvention preferably provides a method of degrading trichloroethylene(TCE), other halogenated hydrocarbons which may be degraded by thepresent method include, but are not limited to, tetrachloroethane,tetrachloroethylene (PCE), trichloroethane, dichloroethane (DCA), andchloroform.

The preferred halogenated hydrocarbon compound of the present invention,TCE (1,1,2-trichloroethene), is an aliphatic halogenated hydrocarbonwith the chemical structure HClC═CCl₂. TCE is primarily used in industryas a fire-resisting solvent. It can be produced by removal of onemolecule of hydrogen chloride from acetylene tetrachloride with alkali.

Methanotrophic Bacteria

The present invention utilizes methane-oxidizing bacteria to degrade-thehalogenated hydrocarbon-containing compounds described above.Preferably, bacteria of the strain Methylosinus trichosporium OB3b (MtOB3b) are utilized which produce the soluble form of the enzyme methanemonooxygenase (MMO) Methylssinus trichosporium strain OB3b was placed ondeposit with American Type culture Collection (ATCC), Rockville, Md. onFeb. 15, 1995, under ATCC accession number 55658. Othermethane-oxidizing bacteria which produce MMO may be useful in thepresent invention. These other bacteria include, but are not limited to,Methylosinus sporium, Methylocytis parvus and other species of thegenera Methylomonas, Methylbacter and Methylococcus.

Mt OB3b is an obligate type II methanotrophic bacterium which is capableof growing on methane as its sole source of carbon and energy. Thisbacterium was discovered by Whittenbury et al., J. Gen. Microbiol.,61:205-218 (1970). It is a gram-negative rod- or pear-shaped bacteriumthat forms exospores and which is typically found in rosette-shapedclusters of several cells. Mt OB3b colonies on methane media arewhite-yellow in color. Like other type II methanotrophs, Mt OB3bcontains a complete tricarboxylic acid cycle, and utilizes the serinepathway for formaldehyde assimilation. The DNA of Mt OB3b has a G+Ccontent of 62.5 mol-%. Mt OB3b grows at 37° C., but not at 45° C. Growthof Mt OB3b is not stimulated by yeast extract or by other multi-carboncompounds tested by Whittenbury et al., supra. Mt 0B3b is motile withpolar tuffs of flagella. Capsules formed by Mt OB3b consist of shortfibers radiating from the cell wall and do not respond to polysaccaridestain. The Mt OB3b strain used in the present invention was obtainedfrom Professor R. Whittenbury, Warwick University, United Kingdom andhas been deposited with the National Collection of Industrial Bacteria,Aberdeen, Scotland and assigned number NC1B-11131.

Degradation of Halogenated Hydrocarbon Compounds

The present invention provides a method of microbial degradation of ahalogenated hydrocarbon compound. The method comprises contacting thehalogenated hydrocarbon compound, preferably TCE, with an amount of amethane-oxidizing bacterium, preferably Mt OB3b, effective to completelydegrade the halogenated hydrocarbon compound at a rate from about 500 toabout 10,000 μmicromoles per hour per gram of cells. In the method ofthe present invention the methane oxidizing bacterium is cultured undercontinuous culture conditions so as to produce soluble MMO. As usedherein an effective amount of methane oxidizing bacterium is an amountof the bacterium capable of completely degrading TCE at the rates statedherein. Further, as used herein, the phrase "continuous cultureconditions" means that the methane-oxidizing bacterium has been culturedin a continuously replaced medium that is exposed to a continuous flowof a gas mixture comprising air and methane in a ratio of about25:1-1:20, respectively; preferably about 10:1-1:2; and most preferablyabout 2.1:1. Continuous culture and growth parameters are as describedin Cornish, J. Gen. Micro., 130:2565-2575 (1984) with a dilution rate ofcontinuous culture of about 0.1 volumes/volume culture per hour. Theair-to-methane ratio of the gas mixture may be varied, and we have foundthat methane at concentrations between 1-20% of saturation greatlystimulate TCE oxidation.

We have determined that conventional (i.e. shake flask, non-continuous)methods of culturing the methane-oxidizing bacterium utilized in thepresent invention prove less than satisfactory in terms of the extent ofremoval of TCE from media comprising any significant initialconcentration of TCE. More specifically, we have found that at initialTCE concentrations of one ppm or higher, oxidation of TCE ceased atabout 50% removal when Mt OB3b grown by conventional methods (shakeflask) was utilized.

The halogenated hydrocarbon compound to be degraded by the presentmethod is preferably contacted with the methane-oxidizing bacterium inan aqueous media which comprises about 0.10-20 g/l of themethane-oxidizing bacterium, more preferably from about 0.2 to 2.0g/l ofthe methane-oxidizing bacterium is used. The preferred aqueous mediaused in the present invention is referred to herein as Higgins media.The method of preparing Higgins media is disclosed by Cornish et al., J.Gen. Micro., supra. The recipe utilized in the present invention for thepreparation of Higgins media is given in Example 1 below. Higgins mediais classified as a minimal salts nitrate media, and contains about 250μg/l of Cu²⁺. Other aqueous media suitable for use in the presentinvention may include, but are not limited to those described by Pat etal., Int. J. Systematic Bacteriol., 26:226-229 (1976) as well as AMS andNMS minimal media; see Whittenburg et al., J. Gen. Microlial., supra.

The present method provides for complete degradation of a halogenatedhydrocarbon compound, preferably TCE, present at initial concentrationsup to 10,000 micromoles/l and more preferably at concentrations of fromtrace amounts to 1000 micromoles/l. These concentration values representthe initial concentration of the halogenated hydrocarbon compound in asolution comprising the hydrocarbon compound, the bacterium, and theaqueous media. It is to be understood that trace amounts refers to lowerlimits of detection by assay techniques described in the Examplesherein.

The present method achieves rates of TCE degradation of at least 500μmoles per hour per gram of cells with a range of TCE degradation ratefrom about 500-10,000 μmoles per hour per gram of cells; preferably therange is from about 1000-9000 moles per hour per gram of cells. Morepreferably the present method achieves rates of TCE degradation about2000-4000 moles per hour per gram of cells, where "gram of cells" meansgram of methane-oxidizing bacterium cells on a dry weight basis.

Cultivation of Methane-Oxidizing Bacteria

The present invention is further directed to a method of cultivating amethane-oxidizing bacterium which is capable of completely degrading ahalogenated hydrocarbon compound, preferably TCE. The method comprisesculturing the methane-oxidizing bacterium, preferably Mt OB3b, undercontinuous culture conditions in a suitable minimal salts media, such asHiggins media. The continuous culture conditions comprise exposing thebacterium to a continuous-flow gas mixture of air and methane in a ratioof about 25:1-1:20, preferably about 10:1-1:2, and most preferably about2.1:1. Preferably, the gas mixture during degradation of TCE comprisesmethane at a concentration of about 1-20% of saturation. The presentmethod provides that the continuously cultured methane-oxidizingbacterium will comprise an amount of soluble MMO which is effective tocompletely degrade the halogenated hydrocarbon compound. Preferably, theamount of soluble MMO produced in the continuously cultured bacterialcells is from about 5-30% of the weight of the dry bacterial cells.

Methane Monooxygenase

The methanotrophic bacterium Mt OB3b can comprise both a particulate(i.e., membrane-bound) and a soluble form of the enzyme methanemonooxygenase (MMO). Tonge et al., FEBS Letters, 58:293-299 (1975),reported that MMO is located in the particulate fraction of Mt OB3b, andthat MMO could be solubilized from the particulate fraction by treatmentwith phospholipase D, sonication, or Triton X-100. Tonge et al. alsoreported that ascorbate was an effective electron donor substitute forNADH both in crude extracts of Mt OB3b or in its particulate fraction,but that associated CO-binding cytochrome c seemed to be essential forMMO activity. However, Stirling and Dalton, Eur. J. Biochem., 96:205-212(1979), later reported that the MMO in cell-free extracts of Mt OB3bappeared to be soluble, and that it required NAD(P)H as an electrondonor for activity. Further, they reported that ascorbate was not aneffective electron donor for the MMO of Mt OB3b. Stirling et al.,Biochem J., 77:361-364 (1979), also characterized MMO in several waysincluding its substrate specificity when present in crude abstracts ofMt OB3b.

MMO catalyzes the first step in the oxidation metabolism of methane, inwhich O₂ is cleaved and one oxygen atom is inserted into a C-H bond ofmethane to yield methanol. Fox et al., J. Biol. Chem., 263:10553-10556(1988). The reaction stoichiometry of this step is shown below:

    ______________________________________                                        CH.sub.4 + H.sup.+  + O.sub.2                                                            + NADH →                                                                          CH.sub.3 OH +                                                                           H.sub.2 O + NAD.sup.+                         Methane    Nicotinamide                                                                             Methanol  Nicotinamide                                             Adenine              Adenine                                                  Dinucleotide,        Dinucleotide,                                            reduced              oxidized                                      ______________________________________                                    

Fox and Lipscomb, Biochem. & Biophys. Res. Comm., 154:165-170 (1988),incorporated by reference herein provided a purified MMO from Mt OB3band resolved the MMO system of Mt OB3b into three components, all threeof which they found were required for reconstituting methane-oxidizingactivity in vitro. These components were denoted by Fox and Lipscomb asthe reductase, component B, and the hydroxylase, respectively. The cellsof Mt OB3b utilized in the purification process of Fox and Lipscomb weregrown in continuous culture as described by Cornish et al., J. Gen.Micro., 130:2565-2575 (1984). The Mt OB3b thus cultured reproduciblyexpressed the soluble form of MMO, and could be cultured in high yield.

Degradation of Halogenated Hydrocarbons by Purified MethaneMonooxygenase

We have found that purified MMO components oxidize TCE. Accordingly, thepresent invention envisions a method of degrading a halogenatedhydrocarbon compound by continuously culturing a methane-oxidizingbacterium, preferably Mt OB3b, by exposing the bacterium to continuousculture conditions including a continuous-flow gas mixture comprisingair and methane in a ratio of about 25:1-1:20, preferably about10:1-1:2, and most preferably about 2.1:1, and wherein the bacterialthus cultured comprises soluble MMO in an amount from about 5-30% of theweight of the dry bacterium cells; separating the soluble MMO from thecontinuously cultured bacterium cells, purifying the soluble MMO toyield purified components comprising reductase, component B, andhydroxylase; adding an effective amount of the purified components,preferably in a ratio of about 110:13:250 by weight of the threeComponents, respectively, to an aqueous slurry of the halogenatedaliphatic hydrocarbon compound, preferably TCE, to form a mixture; andreacting the mixture for a period of time sufficient to completelydegrade the halogenated aliphatic hydrocarbon compound.

The invention will be further described by reference to the followingdetailed examples.

Example I Preparation of Higgins Media¹

In order to provide a medium for the cultivation of themethane-oxidizing bacterium suitable for use in the present invention,the following solutions were prepared and stored at 4° C.:

    ______________________________________                                        Ingredient:    Volume %:                                                      100x Higgins Salts Solution                                                   NaNO.sub.3     85                                                             K.sub.2 SO.sub.4                                                                             17                                                             MgSO.sub.4.7H.sub.2 O                                                                        3.7                                                            CaCl.sub.2.2H.sub.2 O                                                                        0.7                                                            100x Higgins Phosphate Solution                                               KH.sub.2 PO.sub.4                                                                            53.0                                                           Na.sub.2 HPO.sub.4                                                                           86.0                                                           Adjust this solution to pH 7.0.                                               500x Higgins Trace Metals Solution                                            ZnSO.sub.4.7H.sub.2 O                                                                        0.287                                                          MnSO.sub.4.7H.sub.2 O                                                                        0.223                                                          H.sub.3 BO.sub.3                                                                             0.062                                                          NaMoO.sub.4.2H.sub.2 O                                                                       0.048                                                          CoCl.sub.2.6H.sub.2 O                                                                        0.048                                                          KI             0.083                                                          CuSO.sub.4.5H.sub.2 O                                                                        0.125                                                          Add 1 ml of 1 mM H.sub.2 SO.sub.4 per                                         liter of Trace Metals Solution.                                               1000x Higgins Iron Solution                                                   Ingredient:    Concentration:                                                 FeSO.sub.4.7H.sub.2 O                                                                        1.12 g/100 ml                                                  Add 5 ml of 1 mM H.sub.2 SO.sub.4 per 100 ml                                  of Higgins Iron Solution.                                                     ______________________________________                                    

Ten ml of Higgins Salts Solution, 10 ml of Higgins Phosphate Solution,and 2 ml of Higgins Trace Metals Solution were mixed together per literof media desired. Distilled water was added to make up the final volume.If agar plates were being made, 17 g of purified agar per liter ofliquid media was added. The mixture was autoclaved for 20 minutes withslow exhaust. When the media had cooled sufficiently to pour plates, orprior to inoculation, 1 ml of Higgins Iron Solution was added by filtersterilization per liter and mixed carefully. Higgins media agar plateswere marked with a red stripe.

Example II Continuous Culture of Mt OB3b

A continuous culture of Mt OB3b was performed in which the bacteriumgrew at rate of approximately 10 hours per generation on Higgins mediumprepared as in Example I. The continuous culture was grown in achemestat growth chamber having a 0.185 liter volume. J. Depamphillisand R. Hanson, J. Bacterial., 98:222-225 (1969), incorporated byreference herein.

The apparatus consisted of a water jacketed growth chamber supplied withsterile, warm moist air and a constant supply of medium (See FIG. 3 inwhich arrows indicate the flow of medium air, warmed water and effluent:oxygen was supplied from the atmosphere). Referring to FIG. 3, thealphabetic references describe: (A) chemostat growth flask (B) mediumpump; (C) medium reservoir; (D) constant temperature bath; (E) constanttemperature heater pump; (F) air humidifying chamber; (G) airsterilizing chamber, (H) air pump and (I) culture collection flask.

a. Growth Chamber

This chamber is composed of two parts; a head section (FIG. 4) and abody section (FIG. 5). The head contains two parts, one for medium andone for air, and is fitted tightly onto the body section by a groundglass joint. The body is water jacketed and has an overflow device tomaintain a constant volume in the chamber. The entrance to this overflowduct is shielded by a glass baffle which helps keep the volume in thechamber constant by preventing the slight amount of foam produced bysparging from leaving the overflow. The maximum volume of the growthchamber was 200 ml.

b. Constant Temperature Apparatus

A. B. Braun Thermomix II (Melsungen, Germany) constant temperature waterpump was used to circulate warmed water (30° C.) through the waterjacket of the growth chamber. This warmed water also heats the air whichis passed into the growth flask (See FIG. 3). The Thermomix II issensitive to changes of 0.1° C so the temperature variation is wellwithin the limits of temperature control required.

c. Aeration System

Bubbles of air were used to supply oxygen and aid in the mixing of thebacterial culture. Air was pumped from the atmosphere by a B 2-F ModelAquarium Pump (Eugene G. Danner Mfg. Co.). The air passed first into awash bottle containing 1% HgCl₂ via a sintered glass sparger and thenthrough a warming bath (30° C.) of sterile water contained in a washbottle which was partially submerged in a water bath. The air thenpassed into the growth chamber via a sintered glass gas dispersion tube.

25 ml of Mt OB3b cells were inoculated into 0.185 l of Higgins media andincubated in the growth chamber at 30° C. New media was continuouslyadded and expelled. The continuous air feed was supplied with anaquarium pump and methane from a pressurized tank. The gas mixture wasapplied in a volume ratio of approximately 1:1 (CH₄ :air). The growthvessel was stirred vigorously. Mt OB3b cells were grown to variousturbidities and measured on a Spectronic 20 spectrometer (600 nm) atwhich time two-phase (Headspace) assays were performed.

Example III Assaying for Rate of TCE Degradation 1. Incubation WithoutHeadspace²

General Protocol:

Each culture of Mt OB3b was added to 1.8 ml serum bottle prewarmed to30° C. and sealed with an 11 mm teflon-lined rubber septa. Forcomparisons requiring similar initial dissolved oxygen levels, anaerobicmake-up media (usually 0.6 ml) was added to the bottles first and thenthe bottles were sealed. Air-saturated 30° C. Higgins media prepared asin Example I was added via gas-tight syringe (1.0 ml), while 1 atmpressure was maintained by allowing air to bleed through a 25 gaugeneedle. Finally, culture was added (concentrated to give desireddensity) while remaining headspace-was bled out through the 25 gaugeneedle. TCE was added with a syringe at the bottom of the sealed bottle,with a syringe at top of the bottle removing equivalent volume ofculture. The assay time course was started with TCE (substrate)addition. Incubation was performed at 30° C. with agitation at 200 rpmon a platform shaker.

Assays were terminated by extraction at desired time points. Theliquid-liquid extraction technique used 0.6 ml of pentane containing1,2-dibromoethane as an internal standard added via gas-tight syringe toinverted assay bottle, while a second syringe with a needle below firstneedle level collected displaced solution. Partitioning was brought toequilibrium by centrifugation of bottles at 5000 rpm for 10 min. Theorganic layer was removed by gas-tight syringe and placed in a 1 mlserum bottle for chromatographic analysis. In some cases, dilution ofsample or split injection was necessary. Electron-capture detection waspreferred. The following gas chromatography operating parameters wereused:

                  TABLE I                                                         ______________________________________                                        Gas Chromatography Parameters                                                 ______________________________________                                        GC:         HP5790A (with ECD)                                                Column:     Non-Pakd RSL-160 Thick Film Capillary                                         (Alltech)                                                         Injection   150° C.                                                    Temperature:                                                                  Detection   250° C.                                                    Temperature:                                                                  Ramping:    35° C. (1st min), ramped to 120° C. at                          15° C./min                                                 Carrier Gas:                                                                              H.sub.2                                                           Carrier Gas Flow:                                                                         8 ml/min                                                          Injection Volume:                                                                         1 ml                                                              ______________________________________                                    

No-Headspace Assays of TCE Degradation:

The following Tests 1, 2 and 3 were conducted according to theno-headspace assay procedure described generally above. A summary of theresults of these 3 tests is given in Table II, below:

                  TABLE II                                                        ______________________________________                                        Summary of Results of No-Headspace Assays                                                           Rate of TCE-utilization                                        Culture turbidity                                                                            (μmoles · hr.sup.-1 · g                                  cells.sup.-1)                                           Test   (Absorbence 600 nm)                                                                          (no headspace assay)                                    ______________________________________                                        1      1.310           336                                                    2      1.410          1070                                                    3      1.460          2400                                                    ______________________________________                                    

The exact assay protocol used and the detailed results obtained in eachtest are given below.

Test 1--No-Headspace Assay

Protocol:

2 ml of Mt OB3b cell suspension grown in a chemostat (procedure) withabsorbance A₆₀₀ =1.310 were added to 8 ml of 30° C. Higgins mediaprepared as in Example 1 in prewarmed 120 ml serum bottles (1/5 dilutionA₆₀₀ =0.252). The bottles were evacuated and refilled with air having 0%methane. One bottle was heat-killed and used as a control.

1.79 ml of Mt OB3b cell suspension was added to sealed 1.8 ml serumbottles. 11.25 μof 4 mM TCE stock was added to start the assay with anominal initial TCE concentration of 25 μM. Bottles were sacrificed at2.5, 5.0, 10.0 and 15.0 minutes by displacing 0.6 ml aqueous solutionwith pentane containing 1 ppm 1,2-dibromoethane as an internal standard.

The results of Test 1 are given in Table III, below:

                  TABLE III                                                       ______________________________________                                        Test 1 Results                                                                Time         [TCE], ppm [TCE], μM                                          ______________________________________                                        Run 1 Results:                                                                Heat-killed control, 0% methane:                                               2.5 min     1.836      13.97                                                  5.0 min     2.691      20.48                                                 15.0 min     2.883      21.94                                                 Run 2 Results:                                                                1/5 dilution, 0% methane:                                                      2.5 min     2.103      16.01                                                  5.0 min     0.261      1.99                                                  10.0 min     0.255      1.94                                                  15.0 min     0.204      1.55                                                  ______________________________________                                    

Test 2--No-Headspace Assay

Test 2 was performed following the same procedures described above forTest 1, except that 1/20 and 1/50 dilutions were performed. The resultsof Test 2 are given in Table IV, below:

                  TABLE IV                                                        ______________________________________                                        Test 2 Results                                                                A.sub.600 = 1.410 from chemostat                                              1/20 dilution A.sub.600 = 0.069                                               1/50 dilution A.sub.600 = 0.026                                               Nominal initial TCE concentration = 25 μM                                  Time         [TCE], ppm [TCE], μM                                          ______________________________________                                        Run 1 Results:                                                                Heat-killed control, 0% methane:                                               2.5 min     2.749      20.92                                                  5.0 min     2,861      21.77                                                 10.0 min     2.901      22.08                                                 15.0 min     3.132      23.84                                                 Run 2 Results:                                                                1/20 dilution, 0% methane:                                                     2.5 min     4.227      32.17                                                  5.0 min     3.475      26.45                                                 10.0 min     2.843      21.64                                                 15.0 min     2.742      20.87                                                 Run 3 Results:                                                                1/50 dilution, 0% methane:                                                     2.5 min     2.924      22.25                                                  5.0 min     1.843      14.03                                                 10.0 min     2.717      20.68                                                 15.0 min     3.183      24.22                                                 ______________________________________                                    

Test 3--No-Headspace Assay

In prewarmed (30° C.) 120 ml serum bottles, 1 ml of Mt OB3b cellsuspension grown in a chemostat (A₆₀₀ =1.460) were mixed with 9 mls of30° C. Higgins media prepared as in Example I. One bottle was preparedby adding "spent" media from the chemostat (decant media after pelletingcells with centrifugation--10,000 rpm for 10 minutes) rather than freshHiggins media. The bottles were sealed with 20 mm teflon-lined rubbersepta, evacuated and refilled with air (0% methane). One bottle washeat-killed and used as a control.

1.76 ml of Mt OB3b cell suspension was added to sealed 1.8 ml serumbottles (25 gauge needle used to equilibrate pressure). 45 μl of 4 mMTCE stock were added to start the assay. Bottles were incubated at 30°C. with agitation on a shaker bath. Bottles were sacrificed at 2.5, 5.0,10.0, 15.0 and 20.0 mins by displacing 0.6 ml aqueous solution withpentane containing 1 ppm 1,2-dibromoethane as an internal standard.Analysis was performed on a HP 5790A GC using ECD.

The results of Test 3 are given in Table V, below:

                  TABLE V                                                         ______________________________________                                        Test 3 Results                                                                A.sub.600 = 1.460 from chemostat                                              1/10 dilution A.sub.600 = 0.140                                               Nominal initial TCE concentration = 100 μM                                 (using 4 mM TCE stock in water)                                               Dry weight = 0.10 g · l.sup.-1                                       Run 1 Results:                                                                Heat-killed control, 0% methane:                                              Time, min    [TCE], ppm [TCE], μM                                          ______________________________________                                        2.5          15.679     119.33                                                5.0          14.713     111.98                                                10.0         14.760     112.34                                                15.0         15.230     115.91                                                20.0         14.691     111.81                                                ______________________________________                                                                     Rates                                            Time, min                                                                             [TCE], ppm [TCE], μM                                                                            μmoles · h.sup.-1 · g                                    cells.sup.-1                                     ______________________________________                                        Run 2 Results:                                                                1/10 dilution in Higgins media, 0% methane:                                   2.5     14.119     107.46                                                     5.0     14.651     111.41                                                     10.0    11.981     91.19                                                      15.0    8.406      63.98     3200 (10 min-15 min)                             20.0    7.208      54.86                                                      Run 3 Results:                                                                1/10 dilution in spent Higgins media from chemostat, 0% methane:              2.5     14.277     108.66                                                     5.0     13.757     104.70                                                     10.0    12.924     98.36                                                      15.0    8.543      65.02     3960 (10 min-15 min)                             20.0    7.650      58.22                                                      ______________________________________                                    

The rate at those times in the reactions when most rapid oxidation ofTCE occurred was approximately 3500 μmoles/h⁻¹.g cells⁻¹. This rate wasnot stimulated by the addition of methane to the reaction mixture. Therate of TCE oxidation in the presence of methane over time is linear andTCE oxidation is more complete. TCE oxidation also occurs more rapidlyin the presence of low concentrations of methane (Table VII). Therefore,it is believed that cells can oxidize TCE at rates of 10,000μmoles.h⁻¹.g cells⁻¹ under optimal conditions.

2. Incubation with Headspace

Each culture of Mt OB3b was added in 2 ml quantities to 10 ml serumbottles. Bottles were sealed with 20 mm teflon-lined rubber septa. TCEwas added to start the assay. Headspace accounted for 80% of totalvolume within bottles, but TCE concentration was added based on 2 mlaqueous phase. TCE resided principally in the headspace. All bottleswere inverted in order to prevent possible TCE loss by trapping thecompound in the headspace between the liquid phase and glass. Sampleswere incubated inverted at 30° C. with agitation (200 rpm on a platformshaker).

Preparation of GC Calibration Curves:

Headspace incubations were analyzed by direct injection of headspaceinto a gas chromatograph. This method may be performed withoutsacrificing a sample. It is important to prepare sound calibrationcurves for quantitation. The best method for external standardization isto prepare samples as if running an assay and heat-killing at 80° C. for10 mins prior to TCE addition. Samples were incubated for 30 mins undertest conditions to allow adequate time for TCE to partition among thenumerous phases (air, water, cell material, and the like). Headspacesamples were injected into a gas chromatograph using either FID or ECD.Numerous TCE concentrations were used to obtain a sound standardizationcurve.

Example IV TCE Degradation with Headspace Assay

Headspace assays of TCE degradation were conducted by adding bacterialcells from the continuous culture described in Example II or cellsdiluted with spent Higgins medium (2 mls) into assay vials (10 mlvials).

Dilution of cells in spent Higgins medium had no effect on the rate ofTCE oxidation. Therefore, there do not seem to be protective compoundsin the medium.

Heat-killed controls indicated that no TCE was lost from the vials.

The rates of TCE utilization in two-phase head space assays are shown inTable VI, below. Rates were calculated from peak heights of recordertracings from a gas chromatograph equipped with an electron capturedetector. The gas chromatograph parameters reported in Table I hereinwere utilized.

                  TABLE VI                                                        ______________________________________                                        TCE Degradation in Two-Phase Assay                                                            Initial TCE                                                                              Rate, micromoles                                   Cell density    conc.      TCE oxidized ·                            in assay vials (g · l.sup.-1)                                                        (micromolar)                                                                             g cells.sup.-1 · hr.sup.-1                ______________________________________                                        .695            22         281                                                .521            22         308                                                .347            22         465                                                .173            23         461                                                .070            22         808                                                ______________________________________                                    

Example V Effect of Mt OB3b Cell Density on TCE Degradation Rate

In this example, the cell mass of the continuous culture (generated inExample II) increased to 0.825 g/l from 0.695 g/l. As shown in FIG. 1,the rate of TCE degradation at low cell densities increased to 1200micromoles TCE removed.hr⁻¹.g cells⁻¹ at an initial TCE concentration of80 μM.

The rate of methane oxidation by these cells during culturing was 2860moles.hr⁻¹.g cells⁻¹.

The curves at two different Mt OB3b cell densities shown in FIG. 1 alsoillustrate that TCE oxidation was less complete at low cell densitiesthan at high cell densities. This indicates that cells at high densitieswithstand toxic intermediates because there is more biomass available toreact with the reactive intermediate compounds.

Alternatively, the slower rates of oxidation per unit mass at high celldensities may have limited the rate of production of toxic intermediatesto a rate at which they were further degraded.

Example VI TCE Degradation with Single Phase Assay

In order to test the hypothesis that gas transfer limited oxidationrates at high cell densities, the rate of TCE degradation was assayed ina single phase assay using techniques for no head space assay describedin Example III. The assay was performed at a Mt OB3b cell density ofapproximately 0.160 g/l, and an initial TCE concentration of 80 μmoles.The rate of TCE oxidation was 2400 micromoles.hr⁻¹ g cells⁻¹ or 315mg.hr⁻¹.g cells⁻¹.

Example VII Effect of Methane Present During TCE Degradation

In this example the effect of methane present in the TCE oxidationvessel on the rate of oxidation of TCE was determined. A no head spaceassay of TCE degradation was performed according to the procedures ofExample III in which the Mt OB3b cell density was approximately 0.16g/l, and the nominal initial TCE concentration was 80 μM. The resultsare presented in Table VII, below and FIG. 2.

                  TABLE VII                                                       ______________________________________                                        Rate of TCE Degradation in Presence of Methane                                Methane added to                                                              reaction system,                                                                            Rate of TCE oxidation,                                          (% saturation)                                                                              μmoles · hr.sup.-1 · g                     ______________________________________                                                      cells.sup.-1                                                    none          660                                                              5%           3350                                                            10%           3000                                                            20%           875                                                             50%           110                                                             ______________________________________                                    

We have subsequently observed that some cell batches oxidize TCE at rateof approximately 4000 micro-moles.hr⁻¹.g cells⁻¹ without any methanepresent.

Example VIII Effect of Initial TCE Concentration on TCE Degradation Rate

Mt OB3b cells were continuously cultured under the following conditions:

    ______________________________________                                        Medium:       Higgins (prepared as in Example I)                              Gas flow rates:                                                                             methane 45 ml/min                                                             air 135 ml/min                                                  Culture volume:                                                                             220 ml                                                          Culture       30° C.                                                   temperature:                                                                  ______________________________________                                    

A TCE no head space degradation assay of the type in Example III wasperformed using these cells. The results are shown in Table VIII, below:

                  TABLE VIII                                                      ______________________________________                                        Effect of Initial TCE Concentration on Rate of TCE Oxidation                  Concentration                                                                             micromoles TCE removed · hr.sup.-1 · g          of TCE, μM                                                                             dry cells.sup.-1                                                  ______________________________________                                        10           829                                                              25          1104                                                              50          1194                                                              ______________________________________                                    

These results and the results of other experiments led to the conclusionthat the K_(s) for TCE oxidation is approximately 5 μM.

Example IX Degradation of TCE at Various Initial Concentrations

The cell mass of the continuous culture described in Example II wasgrown to 0.64 g cells.1⁻¹. A TCE no head space degradation assay usingthe procedure described in Example III was performed using these cells.The results are shown below in Table IX.

                  TABLE IX                                                        ______________________________________                                        TCE Degradation at Various Initial TCE Concentrations                         Concentration of                                                                         Micromoles TCE removed, μmoles/min.                             TCE, μm 5-10 min.   10-15 min.                                                                              10-25 min.                                   ______________________________________                                         40        2.96                                                                80        5.69        3.03                                                   320        15.54       10.2                                                   640                              10.48                                        ______________________________________                                    

Example X Degradation of TCE by Purified Methane Monooxygenase

Radiolabelled TCE was employed in order to demonstrate the oxidation ofTCE to products upon incubation with soluble methane monooxygenase inthe presence of reduced nicotinamide adenine dinucleotide (NADH). Thethree components of methane monooxygenase, reductase, component B, andhydroxylase, were purified by the method of Fox and Lipscomb, supra, thedisclosure of which is hereby incorporated by reference. Allpurification procedures were performed at 4° C. The cell paste (100 g)was suspended in 200 ml of 25 mM MOPS, pH 7.0, containing 200 μ M Fe(NH₄)₂ (SO₄)₂.6H₂ O and 2 mM cysteine (Buffer A). The cells weredisintegrated by sonication. The sonicated suspension was centrifuged at48000×g for 60 min. The supernatant was decanted, diluted with anadditional 100 ml of buffer A, and adjusted to pH 7.0. The cell freeextract was immediately loaded onto a fast flow DEAE Sepharose CL-6Bcolumn (40 mm×250 mm) equilibrated with buffer A. The column was thenwashed with 600 ml of buffer A. All MMO components were completelyadsorbed. The MMO components were eluted with a 2000 ml gradient of 0.0to 0.40M NaCl in buffer A. The hydroxylase eluted at 0.075M NaCl. Thetwo additional fractions required for reconstituted hydroxylaseactivity, component B and the reductase, eluted at 0.18M NaCl and 0.27MNaCl, respectively. Enzyme incubation mixtures were conducted in sealed10 ml septum vials, each vial containing the ingredients shown in TableX, below:

                  TABLE X                                                         ______________________________________                                        Enzyme Incubation Mixture Components                                                                Amount/                                                 Ingredient            Concentration                                           ______________________________________                                        3-[N-morpholino]propanesulfonic acid                                                                1.5      ml/                                            (MOPS) buffer.sup.1 at pH 7.5                                                                       25       mM                                             NADH.sup.2            0.1      mM                                             Reductase.sup.3       110      μg                                          Component B           13       μg                                          Hydroxylase           250      μg                                          ______________________________________                                         .sup.1 Sigma Chemical Company, St. Louis, Missouri.                           .sup.2 Nicotinamide adenine dinucleotide, reduced form, Sigma Chemical        Company, St. Louis, Missouri.                                                 .sup.3 Reductase, component B, and hydroxylase are the three components o     methane monooxygenase identified by Fox and Lipscomb, supra.             

105 nmol of uniformly labelled ¹⁴ C-TCE was added to each vial byinjection through the rubber septum above the reaction mixture until afinal TCE concentration of 70 μM was reached. The reaction was allowedto proceed for 2 min at 25° C. and then quenched by the addition ofsulfuric acid (Sigma Chemical Company, St. Louis, Miss.) at pH 2.0.

Following centrifugation to remove precipitated protein, the reactionmixtures were analyzed by high pressure liquid chromatography (HPLC).The HPLC operating parameters employed are shown in Table XI, below:

                  TABLE XI                                                        ______________________________________                                        HPLC Operating Parameters                                                     ______________________________________                                        Column:        Aminex HPX-87H HPLC column                                                    (Bio-Rad)                                                      Mobile phase:  25% acetonitrile (Sigma                                                       Chemical Company, St. Louis,                                                  Missouri) in 0.01 N sulfuric                                                  acid (Sigma Chemical Company,                                                 St. Louis, Missouri)                                           Operating mode:                                                                              isocratic                                                      Rate:          0.3 ml per min                                                 Peak detection:                                                                              Ultraviolet spectroscopy,                                                     210 nm                                                         ______________________________________                                    

This HPLC system resolved authentic standards of various potentialorganic acid products and trichloroacetaldehyde (chloral). Under theenzyme incubation conditions described, 53% of the TCE was converted toHPLC-detectable products. These products were formic acid, glyoxylicacid, dichloroacetic acid, and chloral.

In addition to the above incubations, control incubations were conductedin which each one of the methane monooxygenase components or NADH wereomitted. In all of these control experiments, no products of TCEoxidation were detectable.

Thus, this experiment demonstrated that TCE oxidation is catalyzed bythe three-component methane monooxygenase enzyme system in the presenceof NADH.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

What is claimed is:
 1. A method of degrading a halogenated aliphatichydrocarbon compound, said method comprising:(a) culturing amethane-oxidizing bacterium under continuous culture conditionscomprising exposing said bacterium to a continuous-flow gas mixturecomprising air and methane in a ratio of about 25:1-1:20, respectively,the continuously cultured methane-oxidizing bacterium producing asoluble methane monooxygenase in an amount from about 5-30% of theweight of dry bacterium cells; (b) separating said soluble methanemonooxygenase from the continuously cultured methane-oxidizingbacterium; (c) purifying the separated soluble methane monooxygenase toyield purified components comprising reductase, component B, andhydroxylase; (d) adding each of said purified components to an aqueousmixture of said halogenated aliphatic hydrocarbon compound to form asecond mixture; the amount of each of said purified components, whencombined with the other two components in said second mixture, beingeffective to completely degrade said halogenated aliphatic hydrocarboncompound; and (e) reacting said second mixture under conditions and fora period of time sufficient for said purified components to completelydegrade said halogenated aliphatic hydrocarbon compound.
 2. The methodof claim 1 wherein said gas mixture comprises air and methane in a ratioof about 10:1-1:2, respectively.
 3. The method of claim 2 wherein saidgas mixture comprises air and methane in a ratio of about 2.1:1.
 4. Themethod of claim 1 wherein said bacterium is a member of the genusMethylosinus.
 5. The method of claim 4 wherein said bacterium isMethylosinus trichosporium OB3b.
 6. The method of claim 1 wherein saidpurified components reductase, component B, and hydroxylase are added tosaid aqueous slurry in a ratio of about 110:13:250, by weight,respectively.
 7. The method of claim 1 wherein said halogenatedaliphatic hydrocarbon compound is trichloroethylene.